EVOLUTION & DEVELOPMENT
14:6, 522–533 (2012)
DOI: 10.1111/ede.12005
Evolution of the chelicera: a dachshund domain is retained in the
deutocerebral appendage of Opiliones (Arthropoda, Chelicerata)
Prashant P. Sharma,a,b,∗ Evelyn E. Schwager,b Cassandra G. Extavour,b and Gonzalo Giribeta,b
a
Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA
Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02138,
USA
b
∗
Author for correspondence (email: [email protected])
SUMMARY The proximo-distal axis of the arthropod leg is
patterned by mutually antagonistic developmental expression
domains of the genes extradenticle, homothorax, dachshund,
and Distal-less. In the deutocerebral appendages (the antennae) of insects and crustaceans, the expression domain
of dachshund is frequently either absent or, if present, is
not required to pattern medial segments. By contrast, the
dachshund domain is entirely absent in the deutocerebral appendages of spiders, the chelicerae. It is unknown whether
absence of dachshund expression in the spider chelicera is
associated with the two-segmented morphology of this appendage, or whether all chelicerates lack the dachshund domain in their chelicerae. We investigated gene expression
in the harvestman Phalangium opilio, which bears the ple-
siomorphic three-segmented chelicera observed in “primitive”
chelicerate orders. Consistent with patterns reported in spiders, in the harvestman chelicera homothorax, extradenticle,
and Distal-less have broadly overlapping developmental domains, in contrast with mutually exclusive domains in the legs
and pedipalps. However, unlike in spiders, the harvestman
chelicera bears a distinct expression domain of dachshund
in the proximal segment, the podomere that is putatively
lost in derived arachnids. These data suggest that a tripartite proximo-distal domain structure is ancestral to all arthropod appendages, including deutocerebral appendages. As a
corollary, these data also provide an intriguing putative genetic mechanism for the diversity of arachnid chelicerae: loss
of developmental domains along the proximo-distal axis.
INTRODUCTION
Rieckhof et al. 1997; Casares and Mann 1998; Abu-Shaar
et al. 1999; Wu and Cohen 1999; Dong et al. 2001, 2002;
Rauskolb 2001; reviewed by Angelini and Kaufman 2005).
An interesting spatial reversal of exd and hth expression
domains has been documented as follows: exd is expressed
throughout the legs in pancrustaceans (also termed tetraconates), whereas it is restricted to the proximal part in myriapods and chelicerates; hth is expressed throughout the legs
in myriapods and chelicerates, but is restricted proximally in
Pancrustacea (Abu-Shaar and Mann 1998; Abzhanov and
Kaufman 2000; Prpic et al. 2001, 2003; Inoue et al. 2002;
Prpic and Tautz 2003; Angelini and Kaufman 2004, 2005;
Prpic and Damen 2004; Prpic and Telford 2008; Pechmann
and Prpic 2009). Because onychophoran leg gap gene domains are comparable to those of pancrustaceans (Janssen
et al. 2010), the spatial expression of exd and hth has been
interpreted as a potential synapomorphy for the sister group
relationship of chelicerates and myriapods (termed Paradoxopoda or Myriochelata), a relationship recovered in many
molecular phylogenetic analyses (e.g., Hwang et al. 2001;
Mallatt et al. 2004; Pisani et al. 2004; Mallatt and Giribet
2006; Dunn et al. 2008; von Reumont et al. 2009; Rehm et al.
2011). However, this correlation of leg gap gene domains
The articulated appendages of arthropods have facilitated
the tremendous diversity and evolutionary success of this
phylum. Postulated to have evolved from a polyramous ancestral condition, nearly every part of the arthropod leg has
undergone extensive evolutionary modifications, enabling
adaptations to various ecological niches and environments
(Snodgrass 1938; Cisne 1974; Waloszek et al. 2005). Investigation of genetic mechanisms of leg development, principally in the fruit fly Drosophila melanogaster, has implicated a
suite of four genes that pattern the proximo-distal (PD) axis:
Distal-less (Dll), dachshund (dac), extradenticle (exd), and homothorax (hth). In arthropod walking legs, at least three of
these genes (Dll, dac, and either exd or hth) are expressed in
mutually antagonistic domains. Knockdown of these genes
results in loss of the podomeres (leg segments) patterned
by that particular gene, engendering the moniker, “leg gap
genes” (Dong et al. 2001, 2002; Rauskolb 2001). Dll and dac
pattern distal and medial podomeres respectively; proximal
patterning requires the cofactors exd and hth (Sunkel and
Whittle 1987; Cohen and Jürgens 1989; Mardon et al. 1994;
González-Crespo and Morata 1996; Lecuit and Cohen 1997;
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C 2012 Wiley Periodicals, Inc.
Sharma et al.
remains to be tested in chelicerate and myriapod lineages
other than spiders and millipedes.
In contrast with the walking leg, modified appendages are
associated with modified leg gap gene patterning. For example, the mandible of pancrustaceans and myriapods, and the
maxilla of myriapods are considered gnathobasic (Snodgrass
1938; Popadic et al. 1996, 1998). In these appendages, Dll is
not expressed in a manner consistent with PD axis formation
(Scholtz et al. 1998; Abzhanov and Kaufman 2000; Prpic and
Tautz 2003). Similarly, leg gap gene expression in the thoracopods of some crustaceans, and the antennae of insects and
millipedes, differs from that in the walking legs in that mutually antagonistic domains are not observed (e.g., Dong et al.
2001; Williams et al. 2002; Prpic and Tautz 2003; Angelini
and Kaufman 2004). In the D. melanogaster antenna, hth,
dac, and Dll have overlapping expression domains and the
dac medial domain is not functional (Dong et al. 2002; but
see Angelini et al. 2009 for a case of a function antennal dac
domain in Tribolium castaneum). Comparable expression domains of leg gap genes occur in the antennae of other insects
(Angelini and Kaufman 2004, 2005).
The leg gap genes also play a role in conferring antennal
identity. In D. melanogaster knockdown of hth and Dll results
in antenna-to-leg transformations, and increasing dac expression induces medial leg structures in the antenna (Dong
et al. 2001, 2002). A similar effect of hth knockdown has
been reported in the cricket antenna (Ronco et al., 2008),
but in a hemipteran, hth knockdown resulted in the loss of
the antenna altogether (Angelini and Kaufman 2004). Additionally, Dll knockdown does not result in homeotic transformations in the hemipteran antenna (Angelini and Kaufman 2004). Knockdowns or mutations of some other genes
downstream of the leg gap genes can also result in homeotic
antenna-to-leg transformations (Dong et al. 2002; Toegel
et al. 2009; Angelini et al. 2009).
The chelicerate counterpart of the mandibulate antenna is
the chelicera, the namesake of this class of arthropods. Chelicerae are the anterior-most pair of prosomal appendages
and are generally used for feeding. Homology of the antennae of mandibulates and the chelicerae is based on their deutocerebral innervation and Hox gene boundaries (both are
free of Hox expression; Telford and Thomas 1998; Hughes
and Kaufman 2002). However, investigation of leg gap gene
expression in the appendages of spiders—including both mygalomorphs and araneomorphs—has demonstrated the lack
of a dac domain altogether in the chelicera, as well as broadly
overlapping domains of hth, exd, and Dll (Abzhanov and
Kaufman 2000; Prpic et al. 2003; Prpic and Damen 2004;
Pechmann and Prpic 2009). The similarity of overlapping
expression domains in antennae and chelicerae is remarkable. Given the role of leg gap genes in specifying antennal identity in D. melanogaster (Dong et al. 2001, 2002), it
has been suggested that leg gap gene domain overlap and
Harvestman leg gap genes
523
activity is requisite for specification of cheliceral morphology in chelicerates (Prpic and Damen 2004; Pechmann et al.
2010).
One limitation of this inference is that cheliceral morphology is quite variable. The chelicerae of spiders are comprised
of two segments—the proximal basal segment and the distal fang—and are used for envenomation of prey and/or
manipulation of silk. Labidognathous chelicerae (with the
appendage perpendicular to the AP axis) do not occur outside of Araneomorphae (the group that includes orb weavers
and jumping spiders). Orthognathous (with the appendage
parallel to the AP axis) chelicerae occur in Mygalomorphae
(tarantula-like spiders) and Mesothelae (spiders with a segmented opisthosoma), as well as three related arachnid orders: Amblypygi, Uropygi, and Schizomida (the four form
the clade Tetrapulmonata). The chelicerae of these orders
are not chelate (forming a pincer), but rather shaped as a
jackknife (Fig. 1). Another four lineages—Solifugae, Ricinulei, Pseudoscorpiones, and acariform Acari—bear twosegmented chelicerae that are chelate, resembling a pair of
scissors (acariform mites typically bear two cheliceral articles, but some lineages have a reduced third article, the nature
of which is ambiguous; van der Hammen 1989; Evans 1992;
Shultz 2007). Finally, the “primitive” orders of Chelicerata—
Pycnogonida, Xiphosura, Scorpiones, Opiliones, and the extinct Eurypterida (as well as Palpigradi and the parasitiform Acari)—bear three-segmented chelicerae. In the context of chelicerate phylogeny, the spider chelicera is therefore
a derived structure (Fig. 1). Morphological and phylogenetic
studies have previously suggested that a three-segmented chelicera is the ancestral condition, and thus the two-segmented
morphology would have resulted from the loss of one of the
segments, although this hypothesis has not been tested (e.g.,
Dunlop 1996; Wheeler and Hayashi 1998; Giribet et al. 2002;
Shultz 2007).
The occurrence of a cheliceral type with an extra segment is particularly intriguing in the context of leg gap gene
domain evolution. However, the expression domains of leg
gap genes in chelicerate orders that bear three-segmented
chelicerae are not known. As a consequence, it is difficult to
generalize patterns reported for leg gap genes in spiders to all
chelicerates. For example, the absence of the dachshund domain in the spider chelicera could be associated with the twosegmented morphology of this appendage, implying the loss
of one segment, rather than with the chelicera itself. In order
to test this hypothesis, we examined gene expression of the leg
gap genes in the harvestman Phalangium opilio, which bear
the plesiomorphic three-segmented chelicera, and compared
these to data reported for spiders. We also sought similarities
in gene expression in the spider and harvestman chelicerae
to determine which aspects of PD axis specification are conserved in chelicerates. We show that a dachshund domain is
present in the three-segmented harvestman chelicera and is
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Gene identification and whole mount in situ
hybridization
Fig. 1. Phylogeny of Chelicerata indicating relationships among
orders and diversity of chelicerae. Constituent lineages of spiders (order Araneae) as indicated. Orientation of the Araneomorphae schematic indicates labidognathous chelicera (perpendicular to body). Topology derived from Giribet et al. (2001),
Shultz (2007), and Giribet and Edgecombe (2012).
restricted to the proximal segment of this appendage, which
is putatively lost in spiders.
RNA was extracted from a range of embryonic stages using
Trizol (Invitrogen) and first strand cDNA synthesis was performed using SuperScriptIII (Invitrogen). A developmental
transcriptome of P. opilio was generated by sequencing this
cDNA in a single flowcell on an Illumina GAII platform, using paired-end 150-bp-long reads. Thinning was performed
using 0.0496 as the limit (based on Phred quality scores), and
resulting quality of the thinned reads was visualized FastQC
(http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/).
After thinning, only those terminal bases with a Phred
quality score under 30 were trimmed. Assembly was conducted using CLC Genomics Workbench 4.6.1 (CLC bio,
Aarhus, Denmark). All four genes were present in single
copy and sequences ranged in length from 661 to 1665 bp.
Gene identity was confirmed by protein BLAST (NCBI)
and visual inspection of amino acid alignments of orthologs
across Arthropoda. Sequences of all genes are deposited in
GenBank under accession numbers HE805503-HE805507.
Templates for riboprobe synthesis were generated as described by Lynch et al. (2010): genes were amplified by PCR
using gene-specific primers (GSP) with an added linker sequence (5 -ggc cgc gg-3 for the forward primer end and 5 -ccc
ggg gc-3 for the reverse primer). A T7 polymerase binding
site for antisense or sense probe synthesis was generated in
a second PCR using the forward or reverse GSP and a universal primer binding to the 3 or 5 linker sequence with an
added T7 binding site, respectively. GSPs were designed from
the identified transcriptomic assembly. A list of the primers
used for generating sense and antisense probes is provided in
Table S1. Probe synthesis and in situ hybridization followed
the spider protocols for Cupiennius salei (Prpic et al. 2008).
The staining reactions for detection of transcripts lasted between 20 min and 6 h at room temperature. Embryos were
subsequently rinsed with 1× PBS + Tween-20 0.1% to stop
the reaction, counterstained with Hoechst 33342 (Sigma)
10 μg/ml to label nuclei, postfixed in 4% formaldehyde, and
stored at 4◦ C in glycerol. Embryos were mounted in glycerol and images were captured using an HrC AxioCam, a
Lumar stereomicroscope driven by AxioVision v 4.8.2, and
an AxioImager compound microscope driven by AxioVision
v 4.8.2 (Zeiss, Oberkochen, Germany).
MATERIALS AND METHODS
Embryos
Adults of the synanthropic P. opilio (Arachnida, Opiliones,
Eupnoi, Phalangiidae) were hand collected between 9 PM
and 3 AM from various sites in Weston and Woods Hole
(Falmouth), Massachusetts, USA in May through October
of 2009–2011. Adults were maintained and embryos collected
as previously described (Sharma et al. 2012).
RESULTS
Expression of Po-hth and Po-exd
Po-hth is strongly expressed in the head lobes, the labrum,
all of the appendages, and in the ventral ectoderm of all
segments (Fig. 2, Supporting information Fig. S1). In the
pedipalps and walking legs of early embryos (stage 11), Pohth expression is concentrated in the proximal-most part of
Sharma et al.
Fig. 2. Expression of the Phalangium opilio homothorax gene in
the developing appendages. (A–C) Expression in the chelicera,
pedipalp, and L1, respectively, of a stage 11 embryo. Arrowheads indicate median ring of expression. (D–F) Expression in
the chelicera, pedipalp, and L2, respectively, of a stage 14 embryo. Scale bars for all figures are 50 μm. px: proximal segment
of chelicera; 2nd: secondary article of chelicera; da: distal article of chelicera; fe: femur; pa: patella; ti: tibia; mt: metatarsus;
ta: tarsus.
the appendage, and in a separate and medial ring (Fig. 2, B
and C). This ring of expression coincides with that of Po-exd
(see below), although the Po-hth medial domain is broader.
In the walking legs of older embryos (stage 14), the separate expression domains are less marked; Po-hth is strongly
expressed throughout the proximal-most part of the leg, including the endites, to the tibia (Fig. 2F). In these older
stages, a more distal ring of expression is not observed, in
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525
Fig. 3. Expression of the Phalangium opilio extradenticle gene in
the developing appendages. (A–C) Expression in the chelicera,
pedipalp, and L1, respectively, of a stage 12 embryo. Arrowheads indicate distal ring in the patella of the pedipalp and legs.
(D–F) Expression in the chelicera, pedipalp, and L1, respectively, of a stage 15 embryo. Scale bars for all figures are 50 μm.
Abbreviations as in Fig. 2.
contrast to the spider (the hth-1 paralog; Prpic et al. 2003).
In the pedipalp, Po-hth expression is observed throughout
the appendage, except for a distal portion of the tarsus
(Fig. 2E). In the chelicera, Po-hth is expressed throughout
the appendage, except for the distal terminus, where expression is slightly weaker (Fig. 2, A and D).
Po-exd is expressed in the labrum, all of the appendages,
and in the ventral ectoderm of all prosomal and opisthosomal segments (Fig. 3, Supporting information Fig. S1).
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In the appendages, Po-exd is strongly expressed in the
proximal-most parts of the pedipalps and walking legs, corresponding to the coxa and the endite, and a separate and
distinct ring of expression is observed in the patella of the
walking legs and pedipalp (Fig. 3, B and C). This ring is
retained in older stages, albeit wider and with weaker interconnecting expression in the femur and trochanter (Fig. 3,
E and F). In the chelicera, Po-exd is expressed throughout
the appendage except for the distal terminus; no rings of expression are observed, as in the other appendages (Fig. 3, A
and D).
Expression of Po-dac
Po-dac is expressed in the central nervous system, in several
groups of cells in the head lobes, and in the posterior terminus. In older embryos, Po-dac is expressed in the developing
pleurites of the opisthosoma. Expression is never detected in
the labrum (Supporting information Fig. S2). All six pairs of
prosomal appendages express Po-dac (Fig. 4, A–F). In early
stages (stage 10), expression in all limb buds is similar and
occurs in a medial ectodermal ring (Fig. 4, A–C). In older
embryos (stage 14), the pedipalps and legs express Po-dac in a
domain encompassing the podomeres trochanter and femur
(Fig. 4, E and F). In the pedipalp and legs, Po-dac transcripts
are concentrated in the segmental boundaries delimiting the
femur, with slightly weaker interconnecting expression in the
femur (Fig. 4, E and F). Weak expression is observed more
proximally to the coxa; in spiders, this expression is associated with neural structures (Prpic and Damen 2004).
The chelicerae consistently express Po-dac as the appendage elongates (Fig. 4, D, Supporting information Fig.
S2). In early stages (stage 10), strong expression occurs in the
medial part of the cheliceral limb bud ectoderm, in a domain
highly comparable to other limb buds (Fig. 4A). This domain
does not include any part of the body wall. In older embryos,
strong expression of Po-dac is retained in the part of the
chelicera that corresponds to the proximal segment, and no
expression is detected in the secondary or distal articles (Fig.
4D). Contrary to the other leg gap genes, the distal and proximal boundaries of Po-dac expression in the chelicera appear
sharp rather than diffuse. Herein we consider an expression
boundary whose edge is straight and clear to be “sharp” (see,
e.g., Fig. 4, A–C) and otherwise to be “diffuse” (see, e.g.,
Fig. 2, A–C).
Expression of Po-Dll
Po-Dll is expressed in all six prosomal appendages, as well
as in the developing labrum, the posterior terminus, and
the head lobes (Fig. 5, Supporting information Fig. S2).
Unlike spiders, harvestmen do not have any opisthosomal
appendage-derived organs (e.g., spinnerets) and Po-Dll is
Fig. 4. Expression of the Phalangium opilio dachshund gene in
the developing appendages. (A–C) Expression in the chelicera,
pedipalp, and L1, respectively, of a stage 10 embryo. (D–F)
Expression in the chelicera, pedipalp, and L1, respectively, of a
stage 14 embryo. Scale bars for all figures are 50 μm. cx: coxa;
tr: trochanter. Other abbreviations as in Fig. 2.
not expressed in the opisthosoma in a manner suggestive
of rudimentary opisthosomal limb buds. In older embryos
(stage 14), a pair of strong expression domains is observed
in the head lobes, specifically in the part of the eye fields that
coalesce toward the midline during development (Supporting information Fig. 2G). An additional and smaller pair
of expression domains is observed in the head, slightly posterior and lateral to the first pair (Supporting information
Fig. 2G). Expression is also observed in the neuroectoderm
along the ventral midline (Supporting information Fig. 2G),
Sharma et al.
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527
Fig. 5. Expression of the Phalangium opilio Distal-less gene in the developing appendages. (A–C) Expression in the chelicera,
pedipalp, and L1, respectively, of a stage 9 embryo. (D–I) Expression in the chelicera, pedipalp, and L1–L4, respectively, of a stage
14 embryo. Note the expression in the outgrown endites of the pedipalp and first leg (arrowheads), and the lack of these in the other
legs. Scale bars for all figures are 50 μm. Abbreviations as in Fig. 2.
comparable to that in a xiphosuran (Fig. 3J of Mittmann
and Scholtz 2001).
In the pedipalps and legs of early embryos (stage 9), PoDll is strongly expressed in the distal part of the limb bud
(Fig. 5, B, C). Subsequently, Po-Dll appears throughout the
leg as strong rings of expression occurring coincidently with
the developing boundaries of the podomeres, while retaining
strong expression in the distal-most podomere (the tarsus;
Fig. 5, F–I). In older embryos (stage 14), Po-Dll is additionally expressed in the outgrowing endites of the pedipalps and
first walking legs (denoted L1), but not in L2–L4 (Fig. 5,
E–I).
In early stages (stage 9), Po-Dll is expressed in the distal
part of the cheliceral limb bud, comparably to the pedipalps
and walking legs (Fig. 5A). This expression pattern is maintained upon the differentiation of the distal podomere into
the secondary and distal articles (stage 14). Po-Dll continues
to be expressed mostly in the distal part of the appendage,
which will form the second and distal articles; and tapering
expression is observed extending into the proximal segment
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(Fig. 5D). In contrast to the sharp expression boundaries in
the other appendages, the proximal expression boundary of
Po-Dll in the chelicera is diffuse.
DISCUSSION
Here we examined gene expression of the single-copy orthologs of the leg gap genes in the harvestman appendages.
We observe that proximal PD axis patterning of the appendages is conserved in Opiliones and Araneae, and resembles the patterning observed in a glomerid millipede.
These data are consistent with the Myriochelata hypothesis. Second, we report novel expression domains of Dll in
apomorphic structures of harvestmen, namely the outgrown
endites that form the stomotheca and the portion of the eye
fields that form the ocularium. Most significantly, in the harvestman chelicera, the genes hth, exd, and Dll have broadly
overlapping expression domains, as in the spider chelicera
and the mandibulate antenna, but these are independent of
the retention of a dac domain, which patterns the proximal
segment of the harvestman chelicera.
Proximal patterning in harvestmen legs is
consistent with the Myriochelata hypothesis
The expression domains of the leg gap genes hth and exd in P.
opilio are comparable, but not identical, to those of spiders. In
older stages, Po-hth is expressed continuously throughout the
appendage, from the coxa and endite to a distal podomere,
such as the tibia (P. opilio legs) or the tarsus (P. opilio pedipalps). This expression domain approximates that of the spider paralog hth-1, which is similarly broadly expressed from
proximal-most segments to part of the tarsus in the pedipalps and walking legs (Prpic and Damen, 2004; Pechmann
and Prpic 2009). A second paralog common to spiders, hth-2,
is expressed in multiple rings and is believed to be involved
in leg segmentation (Prpic et al. 2003; Pechmann et al. 2009),
but such rings corresponding to podomere boundaries were
not observed in P. opilio. Some lineage-specific differences
exist between the expression domains of Po-hth and spider
hth-1. For example, a separate and more distal ring of hth
expression is not observed in P. opilio, but has been reported
for the hth-1 paralog of spiders (Prpic et al. 2003; Prpic and
Damen 2004). The pedipalps and the walking legs of P. opilio
also have differing distal boundaries of hth expression, unlike
spiders. The significance of these differences is not known.
In contrast to hth, Po-exd is restricted to the proximal
segments and a discrete ring of expression in the patella,
which closely resembles the expression domain of the exd-1
paralog in multiple spider species (Abzhanov and Kaufman
2000; Prpic et al. 2003; Prpic and Damen 2004; Pechmann
and Prpic 2009). In the millipede Glomeris marginata, exd is
similarly expressed in the legs, albeit without the medial ring
domain (Prpic and Tautz 2003).
In spite of these lineage-specific differences, the expression
domains of Po-hth and Po-exd are comparable to those of spiders and a millipede (Abzhanov and Kaufman 2000; Prpic
et al. 2003; Prpic and Tautz 2003; Prpic and Damen 2004;
Pechmann and Prpic 2009). In general, hth is expressed
broadly in much of the developing appendage, whereas exd
is restricted to the proximal podomeres. Taken together
with the inverse spatial relationship of hth and exd in onychophorans and pancrustaceans (Prpic et al. 2003; Prpic and
Telford 2008; Janssen et al. 2010), the expression data observed in P. opilio are consistent with a sister relationship of
chelicerates and myriapods.
The Myriochelata hypothesis is controversial, owing to
discordance with morphological and paleontological data,
as well as numerous phylogenetic and phylogenomic studies that have recovered chelicerates as sister to the remaining arthropods (e.g., Giribet et al. 2001; Regier et al. 2008,
2010). However, other studies, some with deeper gene sampling, have recovered the monophyly of chelicerates and myriapods (Hwang et al. 2001; Mallatt et al. 2004; Pisani et al.
2004; Mallatt and Giribet 2006; Dunn et al. 2008; Hejnol
et al. 2009; von Reumont et al. 2009; Rehm et al. 2011). Consequently, although the spatial relationship of hth and exd
in arthropods constitutes a poorly sampled, one-character
system, it is plausible that PD axis patterning in myriapod
and chelicerate appendages constitutes a homologous condition. Myriochelata is also supported by detailed similarities
in chelicerate and myriapod neurogenesis (Dove and Stollewerk 2003; Kadner and Stollewerk 2004; Mayer and Whitington 2009), which contrasts with the neuroblast-driven system
present in insects and crustaceans (Ungerer et al. 2011).
A role for Dll in patterning harvestman
apomorphies
Consistent with its role in patterning outgrowths, Dll is expressed in the distal parts of all appendages. Additional expression domains occur in the labrum and telson, which have
been reported in various other arthropod species (e.g., Panganiban et al. 1995; Popadic et al. 1998; Thomas and Telford
1999; Abzhanov and Kaufman 2000). Like the other leg gap
genes, Dll is known to have additional roles in development
beyond the PD axis, such as patterning sensory organs and
bristles (Sunkel and Whittle 1987; Cohen and Jürgens 1989;
Mittmann and Scholtz 2001; Williams et al. 2002), and even
gap gene function in spiders (Pechmann et al. 2011). Here
we observed two additional domains of Dll function that are
unique to the harvestman.
First, Dll is expressed in the endites of both the pedipalps and the first walking legs. These domains of expression
are similar to the Dll expression domains in the endites of
Sharma et al.
crustaceans (Panganiban et al. 1995). In P. opilio, the endites
of these two appendages elongate in the adult, forming a preoral cavity called the stomotheca—a structure that occurs
only in harvestmen and scorpions, and the putative synapomorphy of this clade (Stomothecata sensu Shultz 2007;
Fig. 1). The other endites of the harvestman neither elongate
nor express Dll (Fig. 5, E–I). In the spider, the pedipalpal
endite expresses Dll, but other endites do not (Schoppmeier
and Damen 2001; Prpic and Damen 2004; Pechmann and
Prpic 2009). As in the harvestman, the Dll-expressing endite
of spiders is retained in the adult, forming the spider’s “maxilla” (not homologous to the mandibulate maxilla). Taken
together, these data suggest that Dll is involved in patterning
the endites that form gnathobasic mouthparts in chelicerates.
Expression data from mouthparts of scorpions, which could
further test this hypothesis, are not presently available.
Second, Dll is expressed in a pair of domains in the center
of the each eye field. Dll expression in the head lobes has been
observed in other chelicerates, but Dll expression in spider
and mites is either peripheral or diffuse, in comparison to
the harvestman (Thomas and Telford 1999; Abzhanov and
Kaufman 2000; Schoppmeier and Damen 2001; Pechmann
and Prpic 2009). Moreover, the pair of domains that strongly
express Dll in P. opilio subsequently form a fused outgrowth
called the ocularium, a stalk-like structure that bears a single
pair of simple ocelli, in the adult (Juberthie 1964). A similar
eye mound also occurs in pycnogonids, but as with scorpions, expression data for the pycnogonid eye mound are not
presently available. As with the endites, the co-occurrence
of the expression domains and subsequent outgrowth in the
locality of the expression suggest that Dll is involved in ocularium formation.
Functional tests of Dll activity by dsRNAi-mediated
knockdown have been conducted in a spider and in a mite
(Schoppmeier and Damen 2001; Khila and Grbic 2007). In
the mite, the knockdown is reported to result in truncation
of the pedipalpal endite (Khila and Grbic 2007), whereas in
the spider, the effect of the knockdown on the pedipalpal
endite (or maxilla) was not specified, but this structure is apparently lost as well (Fig. 4, B and D of Schoppmeier and
Damen 2001). Functional methods to test Dll activity in the
endites and ocularium of harvestmen are not yet developed,
but are of significant interest, given other reported cases of
Dll cooption to form nonappendage structures (e.g., butterfly wing spots, McMillan et al. 2002; beetle horns, Moczek
and Rose 2009).
A dac domain is present in the three-segmented
chelicera
In spiders, dac is initially not observed in the two-segmented
chelicera, but is expressed proximally and within the appendage in older stages of C. salei (Abzhanov and Kaufman
Harvestman leg gap genes
529
2000; Prpic and Damen 2004; Pechmann and Prpic 2009).
This late-stage expression was previously postulated to be
of neural nature, as comparable expression also occurred in
the coxae of all other appendages of older C. salei embryos
(Prpic and Damen 2004). In that study, Prpic and Damen
(2004) observed that the broadly overlapping domains of
hth, exd, and Dll in the spider chelicera resembled the expression of these genes in the antenna of D. melanogaster. It
was also conjectured that the lack of antagonistic hth, exd,
and Dll domains in the spider chelicera was associated with
complete loss of the cheliceral dac domain.
Unlike the chelicerae of spiders, the plesiomorphic, threesegmented chelicera of the harvestman expresses dac in a
manner consistent with PD axis patterning during early development. In early embryos, the dac domain in the chelicera is topologically indistinguishable from that in the other
appendage types. However, as the distal portion of the appendage forms an asymmetrical chela, dac is consistently and
strongly expressed in the proximal portion of the chelicera.
Even after the chelicera has formed the three constituent
segments, dac is expressed strongly throughout the proximal
segment. This may imply that the segment missing in the
spider chelicera is the proximal-most one, which is consistent with traditional hypotheses of chelicera evolution based
upon morphology (Dunlop 1996; Wheeler and Hayashi 1998;
Shultz 2007).
Although we do not currently have the tools to test functionally putative mutual antagonisms of the leg gap genes in
the harvestman chelicera, we observe that the expression domains of Po-hth, -exd, and -Dll are broadly overlapping in this
appendage in spite of the presence of a dac domain, much
like their corresponding orthologs in the spider chelicera.
These data suggest that the lack of antagonistic domains
of hth, exd, and Dll in the spider chelicera is not associated with the absence of the dac domain, but rather with the
specification of chelicerae and antennae generally. If these
broadly overlapping domains are involved in conferring cheliceral identity—as with the D. melanogaster antenna—mild
knockdown phenotypes of one or more of these three genes
could result in chelicera-to-leg transformations. Future work
could examine this testable hypothesis, taking advantage of
functional genetic tools available in spiders (Hilbrant et al.
2012).
The retention of dac in the three-segmented chelicera
is remarkable, insofar as dac also occurs in homologous
appendages (the antenna) of some insects, such as D.
melanogaster (Dong et al. 2001), Oncopeltus fasciatus (Angelini and Kaufman 2004), T. castaneum (Prpic et al. 2001),
and Gryllus bimaculatus (Ronco et al. 2008), but not other panarthropods, such as the isopod Porcellio scaber (Abzhanov
and Kaufman 2000). dac is also not observed in the frontal
appendage or jaw of the onychophoran Euperipatoides
kanangrensis (Janssen et al. 2010). Intriguingly, a large dac
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EVOLUTION & DEVELOPMENT
Vol. 14, No. 6, November–December 2012
Fig. 6. Alternative hypotheses of chelicera evolution and summary of known leg gap gene expression domains in chelicerae throughout
Chelicerata. (A) From a three-segmented ancestral state, a two-segmented chelicera could be obtained by loss of the proximal [P],
secondary [S], or the distal [D] segment. If dac is considered a marker of the proximal segment, expression data from spiders and the
harvestman support a transition of chelicera types by loss of the dac domain, and therefore of the proximal segment. (B) Fragmentary
leg gap gene expression data are available for the chelicerae of Chelicerata. The complete suite of expression domain is known only
for opisthothele spiders and harvestmen. For Xiphosura and Acariformes, only the Dll domains have been reported, and are not
depicted.
domain comparable that of P. opilio has only been observed
in one other arthropod: the millipede G. marginata (Prpic and
Tautz 2003). The relevance of this observation to the Myriochelata hypothesis cannot be assessed given the presently
limited data on deutocerebral dac domains in myriapods and
basal chelicerate orders, but is a matter of interest for future
investigation.
The presence of the dac domain in some insects has been
interpreted as a possible retained rudiment of an ancestral tripartite domain structure (Prpic and Damen 2004).
Sharma et al.
However, labile deployment of the leg gap genes in modified appendages precludes the assignment of homology of
structures on the basis of gene expression domains alone
(Williams 1998; Abzhanov and Kaufman 2000). Nevertheless, our observation that a dac domain occurs in the medial
portion of the deutocerebral appendage in a plesiomorphic
order of chelicerates—in addition to a myriapod and some
pancrustaceans—lends credibility to the hypothesized tripartite domain structure of this appendage in the common
ancestor of arthropods, with subsequent losses of particular domains upon modification (as have occurred in other
modified appendage types, such as mandibles and maxillae;
Scholtz et al. 1998; Abzhanov and Kaufman 2000; Angelini
and Kaufman 2005). However, it is also intriguing that during later developmental stages, both the cofactors hth and
exd are expressed continuously and more distally than the
dac domain in the harvestman chelicera. To our knowledge,
the chelicera of P. opilio constitutes the first arthropod appendage wherein this phenomenon occurs, discording with
patterns previously observed for appendage regionalization
via the leg gap genes (Kojima 2004; Angelini and Kaufman
2005).
Comparative functional data are limited for dac, but activity in the deutocerebral appendage appears to vary among
species. For example, in D. melanogaster, the antennal dac
domain is small, limited to the third antennal segment (Mardon et al. 1994). Null dac mutants bear fusion of the a5-arista
joint, but not the loss of any segments, whereas overexpression of the dac domain results in medial leg structures in
the antenna (Dong et al. 2001, 2002). Similarly, dac is weakly
expressed in the proximal antenna of O. fasciatus, and knockdown of dac has no observable effect on the antenna at all
(Angelini and Kaufman 2004). By contrast, despite modest
antennal expression levels (Prpic et al. 2001), knockdown
of dac in T. castaneum induces truncation of the antenna,
owing to the reduction of funicle (medial) segments and fusion of antennal segments in this region, as well as homeotic
transformation of the distal funicle articles toward a club-like
(distal) identity (Angelini et al. 2009). Thus, in the deutocerebral appendage of at least one arthropod lineage, dac acts as
a leg gap gene, as well as confers segmental identity along
the PD axis.
In the harvestman, dac is initially strongly expressed in the
median portion of the cheliceral limb bud, and this domain
is later constrained to the part of the appendage that forms
the proximal segment in P. opilio. Definitive determination
of the role of dac in harvestmen must await the development
of functional genetic tools for this system. However, one intriguing possibility is that, if the cheliceral dac domain of P.
opilio functions in a manner similar to that of T. castaneum,
a knockdown of this gene may result in the loss of the proximal segment, and therefore, in a two-segmented chelicera—
the condition that occurs in derived arachnid orders, such
Harvestman leg gap genes
531
as spiders and other tetrapulmonates (Fig. 6A). Such an experimental result, if tested among several major lineages of
Chelicerata, would support a clear mechanism for the evolutionary transition from the three-segmented chelicera to
the two-segmented types: loss of the dac domain along the
proximo-distal axis.
However, it is presently unknown whether different lineages of chelicerates with two-segmented chelicerae (e.g.,
solifuges, pseudoscorpions, amblypygids) pattern this appendage in the same way (Fig. 6B). Phylogenetic approaches
have previously coded the two- and three-segmented chelicera as two to three separate character states, presuming
homology among these types (Shultz 1990, 2007; Wheeler
and Hayashi 1998; Giribet et al. 2002). It remains to be tested
whether a two-segmented chelicera can be obtained by alternative modifications of the three-segmented ancestral state,
that is by deletions of the second or the distal articles, as
alternatives to the proximal article. A survey of leg gap gene
expression across Chelicerata, with emphasis on dac, may aid
in testing the hypothesis of multiple cheliceral dac domain
losses in derived arachnids as a mechanism for transition to
the two-segmented chelicera.
CONCLUSION
The ancient history and plesiomorphic morphology of harvestmen, here represented by P. opilio, lend itself to investigation of many aspects of early arthropod evolution. We
observed that dac is expressed in the proximal segment of the
chelicera in the harvestman, whereas neither the dac domain
nor this segment is retained in spiders. This correlation suggests that cheliceral segment number is determined by the
presence of the dac domain, providing a putative mechanism
for the evolutionary transitions in chelicera morphology.
Acknowledgments
We are indebted to Sónia C.S. Andrade, Ana Riesgo, and Alicia
Pérez-Porro for technical assistance with the transcriptome of P.
opilio. Dave Smith and Bernhard Götze at the Harvard Center for
Biological Imaging facilitated use of microscopes. Comments from
Elizabeth L. Jockusch and Frank Smith refined some of the ideas
presented. EES was supported by DFG fellowship SCHW 1557/11. This work was partially supported by NSF grant IOS-0817678 to
CGE, and by internal MCZ funds to GG. Comments from editor
Lisa M. Nagy and two anonymous reviewers improved an earlier
version of the manuscript.
REFERENCES
Abu-Shaar, M., and Mann, R. S. 1998. Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg
development. Development 125: 3821–3830.
Abu-Shaar, M., Ryoo, H. D., and Mann, R. S. 1999. Control of the
nuclear localization of extradenticle by competing nuclear import
and export signals. Genes Dev. 13: 935–945.
532
EVOLUTION & DEVELOPMENT
Vol. 14, No. 6, November–December 2012
Abzhanov, A., and Kaufman, T. C. 2000. Homologs of Drosophila appendage genes in the patterning of arthropod limbs. Dev. Biol. 227:
673–689.
Angelini, D. R., and Kaufman, T. C. 2004. Functional analyses in the
hemipteran Oncopeltus fasciatus reveal conserved and derived aspects
of appendage patterning in insects. Dev. Biol. 271: 306–321.
Angelini, D. R., and Kaufman, T. C. 2005. Insect appendages and comparative ontogenetics. Dev. Biol. 286: 57–77.
Angelini, D. R., Kikuchi, M., and Jockusch, E. L. 2009. Genetic patterning in the adult capitate antenna of the beetle Tribolium castaneum.
Dev. Biol. 327: 240–251.
Casares, F., and Mann, R. S. 1998. Control of antennal versus leg development in Drosophila. Nature 392: 723–726.
Cisne, J. L. 1974. Evolution of the world fauna of aquatic free-living
arthropods. Evolution 28: 337–366.
Cohen, S. M., and Jürgens, G. 1989. Proximal-distal pattern formation
in Drosophila: Graded requirement for Distal-less gene activity during
limb development. Wilhelm Roux’s Arch. Dev. Biol. 198: 157–169.
Dong, P. D. S., Chu, J., and Panganiban, G. 2001. Proximodistal domain
specification and interactions in developing Drosophila appendages.
Development 128: 2365–2372.
Dong, P. D. S., Dicks, J. S., and Panganiban, G. 2002. Distal-less and homothorax regulate multiple targets to pattern the Drosophila antenna.
Development 129: 1967–1974.
Dove, H., and Stollewerk, A. 2003. Comparative analysis of neurogenesis in the myriapod Glomeris marginata (Diplopoda) suggests more
similarities to chelicerates than to insects. Development 130: 2161–
2171.
Dunlop, J. A. 1996. Evidence for a sister group relationship between
Ricinulei and Trigonotarbida. Bull. Br. Arachnol. Soc. 10: 193–
204.
Dunn, C. W., et al. 2008. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452: 745–749.
Evans, G. O. 1992. Principles of Acarology. CABI, Wallingford, UK.
Giribet, G., and Edgecombe, G. D. 2012. Reevaluating the arthropod
tree of life. Annu. Rev. Entomol. 57: 167–186.
Giribet, G., Edgecombe, G. D., and Wheeler, W. C. 2001. Arthropod
phylogeny based on eight molecular loci and morphology. Nature
413: 157–161.
Giribet, G., Edgecombe, G. D., Wheeler, W. C., and Babbitt, C. 2002.
Phylogeny and systematic position of Opiliones: A combined analysis
of chelicerate relationships using morphological and molecular data.
Cladistics 18: 5–70.
González-Crespo, S., and Morata, G. 1996. Genetic evidence for the
subdivision of the arthropod limb into coxopodite and telopodite.
Development 122: 3921–3928.
Hilbrant, M., Damen, W. G. M., and McGregor, A. P. 2012. Evolutionary crossroads in developmental biology: the spider Parasteatoda
tepidariorum. Development 139: 2655–2662.
Hejnol, A., et al. 2009. Assessing the root of bilaterian animals with
scalable phylogenomic methods. Proc. R. Soc. Lond. B 276: 4261–
4270.
Hughes, C. L., and Kaufman, T. C. 2002. Hox genes and the evolution
of the arthropod body plan. Evol. Dev. 4: 459–499.
Hwang, U. W., Friedrich, M., Tautz, D., Park, C. J., and Kim, W. 2001.
Mitochondrial protein phylogeny joins myriapods with chelicerates.
Nature 413: 154–157.
Inoue, Y., et al. 2002. Correlation of expression patterns of homothorax,
dachshund, and Distal-less with the proximodistal segmentation of
the cricket leg bud. Mech. Dev. 113: 141–148.
Janssen, R., Eriksson, B. J., Budd, G. E., Akam, M., and Prpic,
N.-M. 2010. Gene expression patterns in an onychophoran reveal
that regionalization predates limb segmentation in pan-arthropods.
Evol. Dev. 12: 363–372.
Juberthie, C. 1964. Recherches sur la biologie des Opilions. Dissertation,
Université de Toulouse, Toulouse, France.
Kadner, D., and Stollewerk, A. 2004. Neurogenesis in the chilopod
Lithobius forficatus suggests more similarities to chelicerates than to
insects. Dev. Genes Evol. 214: 367–379.
Khila, A., and Grbic, M. 2007. Gene silencing in the spider mite Tetrany-
chus urticae: dsRNA and siRNA parental silencing of the Distal-less
gene. Dev. Genes Evol. 217: 241–251.
Kojima, T. 2004. The mechanism of Drosophila leg development along
the proximodistal axis. Develop. Growth Differ. 46: 115–129.
Lecuit, T., and Cohen, S. M. 1997. Proximal-distal axis formation in the
Drosophila leg. Nature 388: 139–145.
Lynch, J. A., Peel, A. D., Drechsler, A., Averof, M., and Roth, S. 2010.
EGF signaling and the origin of axial polarity among the insects.
Curr. Biol. 20: 1042–1047.
Mallatt, J. M., Garey, J. R., and Shultz, J. W. 2004. Ecdysozoan phylogeny and Bayesian inference: First use of nearly complete 28S and
18S rRNA gene sequences to classify the arthropods and their kin.
Mol. Phylogenet. Evol. 31: 178–191.
Mallatt, J., and Giribet, G. 2006. Further use of nearly complete, 28S
and 18S rRNA genes to classify Ecdysozoa: 37 more arthropods and
a kinorhynch. Mol. Phylogenet. Evol. 40: 772–794.
Mardon, G., Solomon, N. M., and Rubin, G. M. 1994. dachshund encodes a nuclear protein required for normal eye and leg development
in Drosophila. Development 120: 3473–3486.
Mayer, G., and Whitington, P. M. 2009. Velvet worm development links
myriapods with chelicerates. Proc. R. Soc. Lond. B 276: 3571–3579.
McMillan, W. O., Monteiro, A., and Kapan, D. D. 2002. Development
and evolution on the wing. Trends Ecol. Evol. 17: 125–133.
Mittmann, B., and Scholtz, G. 2001. Distal-less expression in embryos
of Limulus polyphemus (Chelicerata, Xiphosura) and Lepisma saccharina (Insecta, Zygentoma) suggests a role in the development of
mechanoreceptors, chemoreceptors, and the CNS. Dev. Genes Evol.
211: 232–243.
Moczek, A. P., and Rose, D. J. 2009. Differential recruitment of limb
patterning genes during development and diversification of beetle
horns. Proc. Natl. Acad. Sci. USA 106: 8992–8997.
Panganiban, G., Sebring, A., Nagy, L., and Carroll, S. 1995. The development of crustacean limbs and the evolution of arthropods. Science
270: 1363– 1366.
Pechmann, M., Khadjeh, S., Sprenger, F., and Prpic, N.-M. 2010.
Patterning mechanisms and morphological diversity of spider appendages and their importance for spider evolution. Arthropod Struct.
Dev. 39: 453–467.
Pechmann, M., Khadjeh, S., Turetzek, N., McGregor, A. P., Damen, W.
G. M., and Prpic, N.-M. 2011. Novel function of Distal-less as a gap
gene during spider segmentation. PLoS Genet. 7: e1002342.
Pechmann, M., and Prpic, N.-M. 2009. Appendage patterning in the
South American bird spider Acanthoscurria geniculata (Araneae: Mygalomorphae). Dev. Genes Evol. 219:189–198.
Pisani, D., Poling, L. L., Lyons-Weiler, M., and Hedges, S. B. 2004. The
colonization of land by animals: Molecular phylogeny and divergence
times among arthropods. BMC Biol. 2: 1–10.
Popadic, A., Panganiban, G., Abzhanov, A., Rusch, D., Shear, W. A.,
and Kaufman, T. C. 1998. Molecular evidence for the gnathobasic
derivation of arthropod mandibles and the appendicular origin of the
labrum and other structures. Dev. Genes Evol. 208: 142–150.
Popadic, A., Rusch, D., Peterson, M., Rogers, B. T., and Kaufman, T.
C. 1996. Origin of the arthropod mandible. Nature 380: 395.
Prpic, N.-M., and Damen, W. G. M. 2004. Expression patterns of leg
genes in the mouthparts of the spider Cupiennius salei (Chelicerata:
Arachnida). Dev. Genes Evol. 214: 296–302.
Prpic, N.-M., Janssen, R., Wigand, B., Klingler, M., and Damen, W.
G. M. 2003. Gene expression in spider appendages reveals reversal of
exd/hth spatial specificity, altered leg gap gene dynamics, and suggests
divergent distal morphogen signaling. Dev. Biol. 264: 119–140.
Prpic, N.-M., Schoppmeier, M., and Damen, W. G. M. 2008. Wholemount in situ hybridization of spider embryos. CSH Protocols 1–4,
doi:10.1101/pdb.prot506.
Prpic, N.-M., and Tautz, D. 2003. The expression of the proximodistal
axis patterning genes Distal-less and dachshund in the appendages of
Glomeris marginata (Myriapoda: Diplopoda) suggests a special role
of these genes in patterning the head appendages. Dev. Biol. 260:
97–112.
Prpic, N.-M., and Telford, M. J. 2008. Expression of homothorax and
extradenticle mRNA in the legs of the crustacean Parhyale hawaiensis:
Sharma et al.
Evidence for a reversal of gene expression regulation in the pancrustacean lineage. Dev. Genes Evol. 218: 333–339.
Prpic, N.-M., Wigand, B., Damen, W. G. M., and Klinger, M. 2001. Expression of dachshund in wild-type and Distal-less mutant Tribolium
corroborates serial homologies in insect appendages. Dev. Genes Evol.
211: 467–477.
Rauskolb, C. 2001. The establishment of segmentation in the Drosophila
leg. Development 128: 4511–4521.
Regier, J. C., et al. 2008. Resolving arthropod phylogeny: Exploring phylogenetic signal within 41 kb of protein-coding nuclear gene sequence.
Syst. Biol. 57: 920–938.
Regier, J. C., et al. 2010. Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature 463:
1079–1084.
Rehm, P., et al. 2011. Dating the arthropod tree based on large-scale
transcriptome data. Mol. Phylogenet. Evol. 61: 880–887.
Rieckhof, G. E., Casares, F., Ryoo, H. D., Abu-Shaar, M., and Mann, R.
S. 1997. Nuclear translocation of extradenticle requires homothorax,
which encodes an extradenticle-related homeodomain protein. Cell
91: 171–183.
Ronco, M., Uda, T., Mito, T., Minelli, A., Noji, S., and Klingler, M.
2008. Antenna and all gnathal appendages are similarly transformed
by homothorax knock-down in the cricket Gryllus bimaculatus. Dev.
Biol. 313: 80–92.
Scholtz, G., Mittmann, B., and Gerberding, M. 1998. The pattern of
Distal-less expression in the mouthparts of crustaceans, myriapods
and insects: New evidence for a gnathobasic mandible and the common origin of Mandibulata. Int. J. Dev. Biol. 42: 801–810.
Schoppmeier, M., and Damen, W. G. M. 2001. Double-stranded RNA
interference in the spider Cupiennius salei: The role of Distal-less
is evolutionarily conserved in arthropod appendage formation. Dev.
Genes Evol. 211: 76–82.
Sharma, P. P., Schwager, E. E., Extavour, C. G., and Giribet, G. 2012.
Hox gene expression in the harvestman Phalangium opilio reveals
divergent patterning of the chelicerate opisthosoma. Evol. Dev. 14:
450–463.
Shultz, J. W. 1990. Evolutionary morphology and phylogeny of Arachnida. Cladistics 6: 1–38.
Shultz, J. W. 2007. A phylogenetic analysis of the arachnid orders
based on morphological characters. Zool. J. Linn. Soc. 150: 221–
265.
Snodgrass, R. E. 1938. Evolution of the Annelida, Onychophora and
Arthropoda. Smithson. Misc. Collns. 97: 1–159.
Sunkel, C. E., and Whittle, J. R. S. 1987. Brista: A gene involved in
the specification and differentiation of distal cephalic and thoracic
structures in Drosophila melanogaster. Wilhelm Roux’s Arch. Dev.
Biol. 196: 124–132.
Telford, M. J., and Thomas, R. H. 1998. Expression of homeobox
Harvestman leg gap genes
533
genes shows chelicerate arthropods retain their deutocerebral segment. Proc. Natl. Acad. Sci. USA 95: 10671–10675.
Thomas, R. H., and Telford, M. J. 1999. Appendage development in embryos of the oribatid mite Archegozetes longisetosus (Acari, Oribatei,
Trhypochthoniidae). Acta Zool. 80: 193–200.
Toegel, J. P., Wimmer, E. A., and Prpic, N.-M. 2009. Loss of spineless
function transforms the Tribolium antenna into a thoracic leg with
pretarsal, tibiotarsal and femoral identity. Dev. Genes Evol. 219: 53–
58.
Ungerer, P., Eriksson, B. J., and Stollewerk, A. 2011. Neurogenesis in the
water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans.
Dev. Biol. 357: 42–52.
Van der Hammen, L. 1989. An Introduction to Comparative Arachnology.
SPB, Leiden, UK.
von Reumont, B. M., et al. 2009. Can comprehensive background knowledge be incorporated into substitution models to improve phylogenetic analyses? A case study on major arthropod relationships. BMC
Evol. Biol. 9: 119.
Waloszek, D., Chen, J., Maas, A., and Wang, X. 2005. Early Cambrian
arthropods—New insights into arthropod head and structural evolution. Arthropod Struct. Dev. 34: 189–205.
Wheeler, W. C., and Hayashi, C. Y. 1998. The phylogeny of the extant
chelicerate orders. Cladistics 14: 173–192.
Williams, T. A. 1998. Distalless expression in crustaceans and the patterning of branched limbs. Dev. Genes Evol. 207: 427–434.
Williams, T. A., Nulsen, C., and Nagy, L. M. 2002. A complex role
for Distal-less in crustacean appendage development. Dev. Biol. 241:
302–312.
Wu, J., and Cohen, S. M. 1999. Proximodistal axis formation in the
Drosophila leg: Subdivision into proximal and distal domains by homothorax and distal-less. Development 126: 109–117.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Fig. S1. Expression of the Phalangium opilio homothorax
and extradenticle genes.
Fig. S2. Expression of the Phalangium opilio dachshund
and Distal-less genes.
Table S1 List of primer sequences used for riboprobe synthesis.